Apamin

Carbachol, GSK1016970A, KB-R7943, HC067047, apamin, and lithium chloride (LiCl) were purchased from Sigma (United States). Iberiotoxin (IbTX), and TRAM34 were purchased from MedChemExpress. The anti-TRPV4 antibody, anti-IP₃R₁ antibody, and KB-R7943 were purchased from Santa Cruz Biotechnology. The anti-NCX₁ antibody, anti-NCX₂ antibody, and anti-NCX₃ antibody were purchased from the Beijing Bioss company.

The cytotoxic effect of BV (melittin comprise approximately 50% and apamin comprise 3% of dried BV) (Chung Jin Biotech Co., Ansan, Korea) [26 (link)], melittin (Enzo Life Sciences AG, Lausen, Switzerland), and apamin (Sigma-Aldrich, St. Louis, Mo, USA) was evaluated using a CellTiter-96^® aqueous cell proliferation assay kit (Promega, Madison, WI, USA). On a 96-well microstate plate, NP fibroblasts were cultured in the presence of 0.1, 1, 3, and 5 μg/mL of BV, 0.1, 1, 3, and 5 μg/mL of melittin, and 0.1, 1, 5, and 10 μg/mL of apamin for 24 h at 37 °C in a 5% CO_2. The reduced tetrazolium compound produces a colored formazan product due to the mitochondrial activity in the cell. The amount of formazan is directly proportional to the number of viable cells. Color intensities were assessed with a fluorescence microplate reader at 490 nm.

The following drugs, acetylsalicylic acid (ASA), capsaicin, capsazepine (CAPZ), l-glutamic acid, phorbol 12-myristate 13-acetate (PMA), bradykinin, yohimbine, pindolol, caffeine, haloperidol, atropine, glibenclamide, apamin, charybdotoxin, tetraethylammonium chloride were procured from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid and dimethyl sulfoxide (DMSO) were procured from Fisher Scientific (Fair Lawn, NJ, USA). All drugs (i.e., bradykinin, capsaicin, l-glutamic acid, and PMA) were dissolved in physiological saline (0.9% [w/v] NaCl), while PECN, ASA, and CAPZ were dissolved in 10% DMSO (v/v). The vehicle had no effects per se on the nociceptive responses in mice when administered alone. The other solutions (i.e., 0.6% Acetic acid) were prepared in 0.9% NaCl. All drugs and chemicals were freshly prepared prior to use and administered in the volume of 10 mL/kg.

The drugs used were the following: DAPA 2–5, DIPA 1–7, DIPA 1–8, DIPA 1–9, DIPA 1–10, and DIPA 1–12 (kindly supplied by Prof. Eddie Wei; Berkeley, CA, USA), TTX (Alomone Labs, Jerusalem, Israel), 5-BT hydrochloride, KCl, carbachol (CCh), tetraethylammonium chloride (TEA), apamin, and IbTX (Sigma-Aldrich, St. Louis, MO, USA). DIPA 1–8, DIPA 1–10, and DIPA 1–12 were dissolved in dimethylsulphoxide (DMSO) (0.1%). Control experiments using DMSO alone did not show any effects on the mechanical activity. All the other drugs were dissolved in distilled water. Chemicals were prepared as stock solution, which were diluted with Krebs solution on the experiment day.

The following drugs were used: (i) acetylsalicylic acid (ASA), apamin, atropine, bradykinin, caffeine, capsaicin, capsazepine (CAPZ), charybdotoxin, glibenclamide, haloperidol, l-glutamic acid, phorbol 12-myristate 13-acetate (PMA), pindolol, tetraethylammonium chloride, and yohimbine were purchased from Sigma-Aldrich (St. Louis, MO, USA); (ii) naltrindole hydrochloride, nor-binaltorphimine dihydrochloride and β-funaltrexamine hydrochloride were purchased from Tocris Bioscience (Ellisville, Missouri, USA); and (iii) acetic acid, dimethyl sulfoxide (DMSO), and methanol were purchased from Fisher Scientific (England). bradykinin, capsaicin, l-glutamic acid, and PMA were dissolved in physiological saline (0.9% (w/v) NaCl), while ASA, MECN, and CAPZ were dissolved in distilled water containing 10% DMSO (v/v). The vehicle used alone had no effects per se on the nociceptive responses in mice. All drugs, chemicals, and MECN solutions were administered in 10 mL/kg volumes and were freshly prepared just before being used.

4-AP, ACh, apamin, EGTA, glibenclamide, iberiotoxin, indomethacin, L-NAME, nifedipine, ODQ, PE, SNP, and verapamil were purchased from Sigma ChemicalCompany (St. Louis, MO, U.S.A.). Charybdotoxin was obtained from Enzo Life Sciences Company (France), and DMSO was obtained from VWR International Ltd. (Prolabo Chemicals, United Kingdom). All substances were dissolved in distilled water except 4-AP, compound 1, glibenclamide, nifedipine, and WL extract, which were dissolved in DMSO and indomethacin in 0.5% w/v Na₂CO₃.